Radiolabelled metal transport proteins as imaging agents

ABSTRACT

A  99mc Tc labeled metal transport protein, products and uses thereof in imaging and especially detecting the presence of high energy/iron uptake tissues such as a tumour within a mammalian body are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a 35 U.S.C. §371 national phase applicationof PCT International Application No. PCT/GB01/03531, having aninternational filing date of Aug. 7, 2001, and claiming priority toGreat Britain Patent Application No. 0019412.6, filed Aug. 8, 2000, thedisclosures of which are incorporated herein by reference in theirentireties. The above PCT International Application was published in theEnglish language and has International Publication No. WO 02/11772 A2.

The present invention relates to a radiolabeled metal transport proteinand use thereof, the radiolabeled metal transport protein being for useparticularly, but not exclusively, in imaging tumour sites within amammalian body.

BACKGROUND TO THE INVENTION

In nuclear medicine, various techniques have been used to visualise thepresence of a tumour within a body. In quantitative terms, technetium(Tc) compounds are by far the most important radiopharmaceuticals usedtoday with an estimated market share of more than 80%.

For radiomedical purposes, the isotope ⁹⁹Tc is important not in itsslowly β-decaying ground state but in a metastable, nuclear excitedstate, i.e. as exclusively γ-emitting ^(99m)Tc with a diagnosticallyuseful half-life of six hours. One of the major reasons for thepopularity of this radioisotope in radiodiagnostics is the availabilityof an easily operable technetium ‘reactor’ or ‘generator’, which allowsthe convenient preparation of applicable solutions in a normal clinicalenvironment.

It is known from the prior art to use the pertechnetate anion[^(99m)TcO₄]⁻ for medical imaging of thyroid disease, based on theprinciple that the pertechnetate anion would behave similarly to iodineand be taken up by the thyroid. The pertechnetate anion has also beenused to image heart, brain, kidney and liver. However, a growing demandfor more specific imaging agents has led to the development ofcovalently linking an appropriate technetium complex to a small peptideor biologically active molecule (BAM). Examples known from the prior artinclude: Tc^(v) complexes linked to bisamidedithiol proligands such asethylenecysteine diester for use in imaging cerebral blood flow in thebrain; ^(99m)Tc-teboroxime and ^(99m)TcN-NOET for imaging the heartdisease; ^(99m)Tc-HIDA, ^(99m)Tc-Lidofenein, ^(99m)Tc-Mebrofenin forimaging the hepatobilary system; ^(99m)Tc-diethylenetriaminepentaaceticacid for imaging kidney disease; and ^(99m)Tc-complexes of phosphonateligands for imaging bone disease. However whilst these compounds aretissue specific none of them is specific for tumour detection.

Further developments in the use of ^(99m)Tc in medical imaging are basedon adapting the outer surface of a technetium complex so as to containgroups necessary for receptor binding. For example, such developmentsinclude labelling progesterone receptors with ^(99m)Tc to identifybreast tumours, labelling central nervous system receptors with ^(99m)Tcto identify psychiatric conditions, epilepsy and Alzheimer's disease andlabelling a variety of antibodies with ^(99m)Tc.

The problem associated with this group of prior art compounds is that,whilst they may be tissue specific, only ^(99m)Tc labelled progesteronereceptors and ^(99m)Tc labelled tumour antibodies can be considered astumour specific imaging agents. Moreover, these compounds are expensive,laborious and difficult to make and often are quite difficult to handle.

A ^(99m)Tc labelled imaging agent that is cell selective, inexpensiveand simple to make would offer immediate advantage over the prior art.

In the present invention we have exploited the characteristics of cellbehaviour and developed a naturally occurring protein which we havelabelled with ^(99m)Tc.

Amongst other characteristics/factors, tumour cells can be distinguishedfrom normal cells by their rapid rate of proliferation. A rapid rate ofcellular proliferation creates a high energy requirement in tumour cellsfor most cellular processes, including a high demand on metaltransportation into the cell by metal transport proteins.

One such group of metal transport proteins are transferrins whichincludes lactoferrin and, being naturally occurring proteins within thebody with a transport function, transferrins inherently transport acrossall membranes, including the blood-brain barrier. The mechanism by whichtransferrins enter the cells and release iron into the cells is asfollows. Circulating transferrin is bound to a specific receptor on thecell surface and is subsequently taken up as a receptor/transferrincomplex by endosomes into the cytosol. Once the receptor/transferrincomplex is in the cytosol the receptor/transferrin complex releases theFe³⁺ or “demetallates” and the apotransferrin and receptor are releasedback out through the cytosol to the cell surface where they may bedegraded, or recycled.

We have used the inherent ability of metal transport proteins to targetcells combined to develop the imaging agents of the present invention.

Accordingly we believe that the present invention, in one aspect,provides tumour-specific imaging agents which will assist the clinicianto make an early clinical diagnosis without the need for invasiveexploratory investigation.

SUMMARY OF THE INVENTION

In its broadest aspect the present invention relates to a ^(99m)Tclabelled metal transport protein and uses thereof in imaging andespecially detecting the presence of a tumour within a mammalian body.

According to a first aspect of the invention, there is provided a^(99m)Tc labelled metal transport protein complex.

Preferably, the metal transport protein is an iron transport protein andmore preferably is a transferrin preferably selected from the groupcomprising lactoferrin, ovotransferrin and/or serum transferrin. Whenthe metal transport protein is a transferrin it is preferentially takenup by tumour cells because of their high energy demand and rate ofproliferation.

Reference herein to transferrin is intended to include lactoferrin,ovotransferrin and/or serum transferrin in their apoprotein ormetal-loaded states.

It will be appreciated that the labelled metal transport protein complexof the present invention carries ^(99m)Tc in the sites normally occupiedby the metal ions. In the embodiment where the metal transport proteinis a transferrin, the protein carries ^(99m)Tc³⁺ in place of Fe³⁺ ionsand encapsulates or folds around the two binding sites occupied by^(99m)Tc³⁺ in a similar way as the transferrin glycoprotein wouldaccommodate Fe³⁺ ions in the natural state. Accordingly, theshape/configuration of the Tc-transferrin is not dramatically distortedor substantially altered from that of the naturally occurringFe-transferrin complex. It is because the ^(99m)Tc-labelled transferrincomplex is similar in structure to endogenous Fe³⁺ carrying transferrinsthat the ^(99m)Tc labelled transferrin complex is likely to berecognised by cells and taken up and processed as an endogenoustransferrin.

Rapidly dividing cells such as tumour cells have high energy andnutrient requirements and have increased demands on a number of normalcellular metabolic processes/activities. Amongst these increasedrequirements is a demand for iron. By utilising the iron metal transportproteins as carriers for ^(99m)Tc, one embodiment of the presentinvention offers a tumour imaging agent that will naturally be attractedto areas within the body that have high iron demand. Similarly, othermetal transport proteins can be used for selectively targetingtissues/sites within the body.

Preferably, the metal transport protein is derived from mammalian tissueor blood and more preferably is a recombinant protein.

Recombinant protein is of particular advantage in that the risk of crosshaematological infection from other factors/agents present in wholeblood, such as by hepatitis virus or HIV, is obviated. Moreover, thereis a current abundance of recombinant lactoferrin that is commerciallyavailable so that a further advantage of the invention resides in thereduced cost of the complex compared to other prior artcompounds/complexes. The recombinant protein may be modified as comparedwith the natural protein, provided that it retains functional metaltransport and receptor binding properties.

According to another aspect of the invention there is provided use of a^(99m)Tc labelled metal transport protein as an imaging agent especiallybut not exclusively, in detecting the presence of a tumour within amammalian body.

Preferably, the ^(99m)Tc labelled metal transport protein furtherincludes any one or more of the preferred features hereinbeforedescribed.

The present invention therefore provides an alternative imaging agentfrom the prior art.

According to a yet further aspect of the invention there is provided aproduct or composition comprising a metal transport protein and areducing agent, the function of the reducing agent being to convert Tcas the pertechnetate (TcO₄ ⁻) to Tc³⁺ so that it may bind to theprotein. In this respect the reducing agent may comprise any agent thatis capable of performing the reduction step.

Preferably, the metal transport protein is as hereinbefore described.

Preferably, the product comprises an amount of metal transport proteinin the range of 2–60 mg.

Preferably, the product or composition further includes a ^(99m)Tcsource, more preferably the source is pertechnetate i.e. TcO₄ ⁻. Thepertechnetate source can be provided with the product or composition ina suitable vial or vessel or it may be provided separately therefrom andadded to the metal transport protein and reducing agent shortly beforeuse. Typically, the pertechnetate source is, provided as a solution;typically it is generated at the site where the investigation/treatmentis to take place.

Preferably, the amount of ^(99m)Tc in the product, when labelled with^(99m)Tc, is in the range of 6–8 GBq and more preferably is about 7.4GBq (200 mCi).

Preferably, the reducing agent is selected from the group comprising atin(II) salt including, for example, chloride, nitrite and/or sulphite.Another prepared reducing agent is ascorbic acid/ascorbate.

Preferably, the product comprises an amount of reducing agent in therange of 0.2–0.3 mg.

Preferably, the product or composition further includes a solubilisingagent and/or an isotonic agent.

Reference herein to an isotonic agent is intended to mean any agentwhich is capable of rendering the composition to an isotonic state insolution with respect to the pH and ionic strength of blood, so thatupon introduction of the product or composition into a body therecipient does not enter a state of shock or suffer any adverse effectto the pH and ionic strength of the composition in solution.

Preferably, the solubilising agent is gentisic acid. The purpose of thesolubilising agent is to enable solubilisation of the Tc³⁺ so as tofacilitate formation of the metal/protein complex, and therefore theexample provided merely illustrates one suitable compound and is notintended to limit the scope of the application.

Preferably, the product comprises an amount of solubilising agent in therange of 0.7–0.9 mg.

Preferably, the isotonic agent comprises a salt and more preferably issodium chloride.

Preferably, the product comprises an amount of the isotonic agent in therange of 20–40 mg.

Preferably the product or composition is lyophilised, that is to say itis freeze dried.

Preferably, the product or composition is pyrogen-free.

Preferably, the product or composition is provided in powder form.

According to a further aspect of the invention there is provided amethod of making a ^(99m)Tc labelled metal transport protein complexcomprising mixing a metal transport protein with a ^(99m)TcO₄ ⁻ sourcein the presence of a reducing agent.

Preferably, the metal transport protein and reducing agent ashereinbefore described are provided in a sealed vial or vessel whichoptionally further includes any one or more of the additiveshereinbefore described.

Prior to administration, a suitable volume of an appropriate aqueoussolution is added to a vial containing the metal transport protein andreducing agent. In practice, the vial is subsequently agitated andallowed to stand for a short period, to ensure that the components havedissolved into the aqueous medium.

Preferably, the ^(99m)TcO₄ ⁻ source is introduced into the resultantaqueous medium as an appropriate aliquot. Alternatively, the ^(99m)Tcsource may be provided with the metal transport protein and/or reducingagent and the aqueous medium as well as any necessary additionalcomponents which can be added thereto. It will be appreciated that thereducing agent converts the pertechnetate into a form of ^(99m)Tc thatis taken up by the metal transport protein so that the resultant complexis labelled.

According to a further aspect of the invention there is provided use ofa metal transport protein for the manufacture of a ^(99m)Tc labelledimaging agent for imaging and especially for diagnosing the presence ofa tumour site within a mammalian body.

According to a further aspect of the invention there is provided use ofa metal transport protein and a reducing agent for the manufacture of animaging agent for imaging and especially for diagnosing the presence ofa tumour site within a mammalian body.

According to a further aspect of the invention there is provided use ofpertechnetate for the manufacture of a ^(99m)Tc labelled metal transportprotein complex imaging agent for imaging and especially for diagnosingthe presence of a tumour site within a mammalian body.

According to a yet further aspect of the invention there is provided amethod of detecting the presence of a tumour within a mammalian bodycomprising the steps of:

-   -   (i) mixing a metal transport protein with an effective amount of        pertechnetate in the presence of a reducing agent and an aqueous        medium so as to produce a ^(99m)Tc labelled metal transport        protein complex,    -   (ii) introducing the resultant aqueous medium into a recipient        under investigation, and    -   (iii) observing images of ^(99m)Tc within the recipient's body        over a selected period.

Preferably, the mixture contains any one or more of the additives and/orfeatures hereinbefore described.

Preferably, the composition in solution is injected into the recipient'sbody, more preferably it is injected by the intravenous route.Alternatively the composition can be taken orally.

According to a yet further aspect of the invention there is provided amethod for detecting a tumour comprising observing images of ^(99m)Tcintroduced into a patient as a labelled metal transport protein complex.

According to a yet further aspect of the invention there is provided useof the method as hereinbefore described for detecting/diagnosing thepresence of a brain tumour.

Reference herein to brain tumour is intended to include any type oftumour or growth which occurs within the blood-brain barrier.

The product or composition and methods of the present invention whichuse transferrins are particularly well suited to the detection of thepresence of a brain tumour in an individual because of the naturaluptake and recognition of the transferrin complex and the inherentability of transferrins to cross the blood-brain barrier. In thisrespect the present invention provides significant advantages over theprior art by provision of a specific tumour-selective imaging agent thatis able to access all areas of brain tissue.

It will be understood from the foregoing statements of invention that^(99m)Tc labelled metal transport protein complexes provide a convenientand cost effective means of detecting the presence of tumours within amammalian body. The present invention in addition provides a method oftreating the tumours once they have been located/identified by using themetal transport protein to carry a radionuclide.

According to a yet further aspect of the invention there is provided amethod of treating a tumour detected using a method or product of theinvention comprising the steps of:

-   -   (i) labelling a metal transport protein with a radionuclide,    -   (ii) producing an aqueous formulation of the radionuclide        labelled metal transport protein, and    -   (iii) introducing the aqueous formulation into a recipient under        treatment.

Preferably, the method further includes the step of, prior to step (i),detecting the presence of a tumour with the ^(99m)Tc labelled metaltransport protein complex of the present invention.

Preferably, the radionuclide labelled metal transport protein complexfurther includes any one or more of the features hereinbefore described.

Preferably, the aqueous formulation of the radionuclide labelled metaltransport protein further includes any one or more of the additiveshereinbefore described.

The method thus provides a means of firstly locating a tumour andsubsequently ensuring that a further dose of a metal transport proteinlabelled with a radionuclide is directed to the same location. Theradionuclide labelled metal transport protein is thus able toattack/target tumour cells specifically. In this way the combinedtherapy can act as a locator and destroyer treatment for tumour cells.As previously mentioned, this combined therapy is envisaged to be ofparticular importance in the diagnosis and treatment of brain tumoursbecause transferrins are able to cross the blood-brain barrier.

Preferably the radionuclide is selected from the group comprising ⁵⁷Co,⁶⁷Cu, ⁶⁷Ga, ⁹⁰Y, ⁹⁷Ru, ¹⁶⁹Yb, ¹⁸⁶Re, ¹⁸⁸Re, ²⁰³Pb, ¹⁵³Sm and/or ²¹²Bi.

According to a yet further aspect of the invention there is provided akit comprising the product or composition of the invention in a suitablysealed vial or vessel and, optionally, a set of written instructions.

According to a yet further aspect of the invention there is provided akit comprising the product or composition of the invention in a suitablysealed vial or vessel and a further product or composition comprising aradionuclide labelled metal transport protein complex in a suitablysealed vial or vessel and optionally, a set of written instructions.

The invention will now be described by way of example only withreference to the following Figures wherein:

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the UV difference spectra for the titration ofapotransferrin with Fe(NTA)₂.

FIG. 2 illustrates a graph representing a titration curve for theaddition of Fe(NTA)₂ to apotransferrin.

FIG. 3 illustrates the dependence of absorbance on time for a solutioncontaining apotransferrin and 2 Molar equivalents Re(NTA).

FIG. 4 illustrates a graph representing the dependence of A₂₃₅ on timefor apotransferrin containing 2 Molar equivalents Re(NTA).

FIG. 5 illustrates the UV difference spectra for titration ofapotransferrin with Re(NTA).

FIG. 6 illustrates a graph representing the reaction of apo-lactoferrinwith Re/NTA,

FIG. 6A showing 1×10⁻⁵ M apo-lactoferrin and 5 mM sodium bicarbonate(black) then with 5-fold excess Re/NTA added after 0 mins, 15, 30, 90,180, 240, 300, and 420 mm.

FIG. 6B illustrates the absorption peak at 231 nm.

FIG. 6C illustrates the 500 nm region in more detail, X representingabsorption peak at 0 and 15 minutes, Y representing maximum absorptionwavelength at the end of the time course.

FIG. 7 illustrates a graph representing the reaction of apolactoferrinand sodium bicarbonate with excess Re/NTA, monitored by measuringabsorbance at 231 nm with time.

FIG. 8 illustrates a graph representing the uptake of the controlSolution 1 (Tc₄ ⁻ and reducing solution) and Solution 2 (TcO₄ ⁻ andreducing solution and Tf).

EXAMPLES DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

Iron containing proteins are known to consist of a single polypeptidechain of around 700 amino acid residues. Both lactoferrin-andtransferrin consist of two lobes, the N-lobe and the C-lobe which haveabout 40% internal sequence homology between them and very similartertiary structure. The folding is identical in the two lobes but the Cterminus lobe contains three extra disulfide bridges, giving it extrastability. Each lobe has a cleft in which the iron atom sits, andbinding induces a conformational change causing the lobe to close overthe metal. Coordination to the metal is by two tyrosine residues, ahistidine and an asparagine. There is also synergistic binding of abidentate carbonate ligand giving an overall six coordinate, distortedoctahedral geometry. The presence of the carbonate is necessary forstrong metal binding. The ligands are attached to different parts of theprotein backbone which allows the lobes to adopt a more open structurein order to release the iron.

Metal binding to transferrin produces new absorption bands in the UV/visspectra, due to the binding to the two tyrosinate residues. UV/visspectroscopy therefore provides a method to distinguish between bindingat the iron site compared to other sites such as platinum binding.UV/vis spectroscopy has also been used in conjunction with titrationexperiments to study the kinetics of binding and to obtain bindingconstants for a variety of metal transferrin complexes. Herein we haveused the same method to determine the binding of rhenium and technetiumto transferrin and lactoferrin.

Tumour cells are known to uptake transferrin at a higher rate thanhealthy cells, and so incorporation of Tc into transferrin andlactoferrin advantageously provides a new method for imaging cancers.Our initial experiments with rhenium whose chemistry is similar totechnetium have led us to the present invention.

Example 1 Metal/Protein Binding

Experimental Procedure

Bovine apotransferrin was washed three times with 0.1 M KCl usingCentricon 30 ultra-filters to remove low molecular mass impurities. Itwas dissolved in 10 cm³ of 10 mM Hepes buffer (pH 7.4) and stored in arefrigerator. Apolactoferrin was prepared from Fe-lactoferrin usingstandard procedures. All glassware was acid soaked for several hoursbefore use to remove any traces of heavy metals and ultrapure water wasused in all experiments. Due to its hygroscopic nature, Re₂O₇ was storedand weighed in a glove box, to ensure accuracy in measurements.

A Perkin-Elmer Lambda 15 spectrophotometer was used for UV/visexperiments, with 10 mM Hepes buffer solution as background correction.

Preparation of Re(NTA)_(n)

NTA refers to nitrilotriacetate, and n is the amount of NTA present insolution relative to rhenium. Re(NTA), where n is equal to 1, wasprepared by adding 4.17 cm³ of NTA solution (0.006 M) to 2.30 cm³ of Reatomic absorption standard solution (1010 μg/ml in 1% HNO₃). Slowaddition of microlitre amounts of 1 M NaOH was carried out to give a pHof between 5 and 6. This solution was then made up to 25 cm³ with 10 mMHepes buffer (pH 7.4), to give an overall rhenium concentration of 0.5mM. Re(NTA)₈ and Re(NTA)₂₀ were made in the same way but using 8.34 cm³and 20.85 cm³ of NTA (0.024 M) respectively.

Preparation of Fe(NTA)₂

A 0.01 M solution of˜Fe(III) nitrate was prepared and 2.50 cm³ wereadded to 16.66 cm³ (2 molar equivalents) NTA solution (0.006 M). Thiswas made up in the same way as Re(NTA) to give a 0.5 mM Fe(NTA)₂solution buffered at pH 7.4 with 10 mM Hepes

UV Spectroscopy

Initially the band at ε₂₈₀93,000 M⁻¹ cm⁻¹ was used to determine theconcentration of washed apotransferrin by UV spectrometry. UV sampleswere then prepared by diluting transferrin or lactoferrin toapproximately 10⁻⁵ M by making the volume up to 3000 μl with 10 mM Hepesbuffer. Immediately before addition of the metal, 60 μl of NaHCO₃ (0.25M) were added to give a 5 mM concentration of bicarbonate.

Titration Experiments

Titration experiments were adapted from those previously carried out inthe literature. Aliquots (usually between 5 and 20 μl) of the Fe(NTA)₂solution were added to the transferrin and the spectrum recorded aftereach addition. This was repeated using aliquots of the Re(NTA)_(n)solutions, but allowing 1.5 hours (or more when appropriate) betweeneach addition to allow equilibration at 310 K.

Time Dependent Spectroscopy

Two molar equivalents of Re(NTA) solution were added to a transferrin orlactoferrin solution and the spectrum recorded at various timeintervals. After equilibrium had been reached two molar equivalents ofFe(NTA)2 were added, and a further spectrum taken.

Reduction of ReO₄ ⁻

0.149 g of Re₂O₇ in 100 cm³ water made a 0.006 M stock solution of ReO₄⁻. 0.5 cm³ of this solution were added to 1 cm³ of SnCl₂ (0.006 M) alongwith 1 cm³ of HCI (1 M). The reduction of the metal was then monitoredby UV spectroscopy, in the presence of varying amounts of NTA. Thisprocess was also repeated using ascorbic acid as the reducing agent inplace of SnCl₂.

These solutions were then used in the same manner as the rhenium atomicabsorption standard, by raising the pH with microlitre amounts of 1 MNaOH and buffering in 10 mM Hepes. UV spectroscopy was used to monitorthe effect of addition to transferrin.

Iron Binding to Transferrin

Upon iron binding the apotransferrin peak at 280 nm decreases in size,while two new peaks appear at 240 and 295 nm which provide a usefuldiagnostic test for metal binding. FIG. 1 shows the UV/vis differencespectra obtained after each addition of Fe(NTA)₂ during the titrationexperiment. The base line of all the UV difference spectra correspondsto apotransferrin before addition of any metal. The increase inabsorbance at 240 and 295 nm is directly related to the percentagesaturation of transferrin binding sites. The increase in absorbance at240 nm was monitored and the change in molar extinction coefficient wasdetermined. The ratio, r, of iron to transferrin was calculated(Table 1) and plotted against the change in extinction coefficient (FIG.2). This shows a relationship between r and Δε at low ironconcentrations (when r is less than one). The slope of the linearportion of the graph was calculated to be 15800±210 M⁻¹ cm⁻¹ whichequates to the molar absorptivity of transferrin with one site saturatedwith Fe. If we assume that the two sites are equivalent, we could expectthe molar absorptivity for transferrin with both sites saturated withiron to be twice the magnitude at approximately 31600 M⁻¹ cm⁻¹. Webelieve these experiments demonstrate that iron binds strongly to thetransferrin and that the transferrin protein remains intact during thesubstitution of iron into the transferrin.

TABLE I Calculation of r and As values for Fe(NTA)2 Volume 10⁻⁶ [Tf]/r([Fe]/ ΔA Δε/M⁻¹ Fe/μl mol dm⁻³ [Tf]) (240 nm) cm⁻¹ 0 1.00 0.00 0.00000 5 9.98 0.08 0.0108 1083 10 9.97 0.17 0.0277 2778 15 9.95 0.25 0.03783803 20 9.93 0.33 0.0533 5369 25 9.92 0.42 0.0624 6293 30 9.9  0.500.0792 7997 40 9.87 0.67 0.1038 10514 50 9.84 0.83 0.1306 13272 60 9.8 1.00 0.1537 15686 70 9.77 1.17 0.1617 16551 80 9.74 1.33 0.1719 17654 909.71 1.50 0.1812 18659 100 9.68 1.67 0.1852 19128 120 9.62 2.00 0.187419478 130 9.58 2.17 0.1903 19865Rhenium Binding to Transferrin

Addition of rhenium to transferrin also showed peaks due to tyrosineπ-π* transitions, at 235 and 292 nm, although the second of these issignificantly smaller in magnitude than for iron. No peak is seen in thevisible region of the spectrum. This is consistent with the spectra seenfor some other metals such as gallium, neodymium, aluminium and indiumwith transferrin.

In contrast to the instantaneous binding shown by iron, the binding ofrhenium to transferrin takes longer to reach an equilibrium. To assessthis, two molar equivalents of Re(NTA) were added to the transferrinsolution, and a spectrum taken at regular time intervals (FIG. 3). FIG.4 shows the increase in absorbance at 235 nm with time and shows that atleast an hour is required for the binding to reach completion.

Under other conditions, such as at temperatures lower than 310 K, orwith more nitrilotriacetate present, the equilibration may take severalhours. Once equilibrium was reached, two molar equivalents of Fe(NTA)were added to the solution and the spectrum obtained is shown by theblue line in FIG. 3. It can be seen that iron has a much faster rate ofbinding and much greater influence on the spectrum. However this slowbinding does not appear to be unusual, and several metals are reportedto require even longer for completion of binding. For example Sc(NTA)₂needs at least 2 h to reach equilibrium and In(NTA)₂ requiresapproximately 6 hours'.

When Fe(NTA)₂ is added to rhenium transferrin (FIG. 3) the change in thespectrum is large and instantaneous. The transferrin has a much greateraffinity for iron than for rhenium and so the rhenium is instantlydisplaced. This also occurs on the addition of iron to many othermetal-transferrins including gallium and scandium.

Rhenium Titration Experiments

Apotransferrin was titrated with Re(NTA), Re(NTA)₈ and Re(NTA)₂₀, andthe UV difference spectra recorded after allowing time for solutions toreach equilibrium (FIG. 5). The absorbance at 235 nm was converted tomolar extinction coefficients, and the ratio, r, of rhenium totransferrin was calculated in the same way as for iron (Table 2).

TABLE 2 Calculation of r and Δε values for Re(NTA)_(n) Volume 10⁻⁶[Tf]/r ([Re]/ ΔA(235 nm) Δε/M⁻¹ cm⁻¹ Re/μl mol dm⁻³ [Tf]) Re(NTA) Re(NTA)₈Re(NTA)₂₀ Re(NTA) Re(NTA)₈ Re(NTA)₂₀ 0 7.35 0.00 0.0000 0.0000 0.0000 00 0 5 7.34 0.11 0.0074 0.0073 0.0069 1012 1000 936 10 7.33 0.22 0.01380.0143 0.0140 1876 1950 1911 15 7.31 0.33 0.0213 0.0209 0.0211 2920 28562889 20 7.30 0.44 0.0279 0.0270 0.0251 3820 3700 3432 25 7.29 0.560.0350 0.0339 0.0292 4800 4650 4004 30 7.27 0.67 0.0423 0.0414 0.03365814 5700 4622 35 7.27 0.78 0.0491 0.0469 0.0362 6760 6450 4979 40 7.250.89 0.0551 0.0506 0.0383 7598 6980 5278 45 7.24 1.00 0.0625 0.05400.0392 8637 7456 5417 50 7.23 1.11 0.0666 0.0578 0.0403 9213 7999 557760 7.21 1.33 0.0729 0.0602 0.0417 10109 8349 5777 70 7.18 1.56 0.07670.0615 0.0430 10678 8563 5993 80 7.16 1.78 0.0795 0.0631 0.0437 111008807 6110 90 7.14 2.00 0.0816 0.0643 0.0454 11435 9000 6356 100 7.112.22 0.0832 0.0664 0.0453 11702 9340 6370 110 7.09 2.44 0.0842 0.06830.0454 11875 9629 6409 120 7.07 2.67 0.0849 0.0701 0.0464 12004 99226565 140 7.02 3.11 0.0887 0.0708 0.0467 12639 10085 6656Rhenium Binding to Lactoferrin

Similar experiments were carried out with lactoferrin and rhenium. Thedata are described below. Re/NTA was then added to the apo-protein andthe absorbance spectra recorded with time, to see if the Re was bound bythe lactoferrin (FIGS. 6(A), (B) and (C) and FIG. 7). Both FIGS. 6(A),(B), (C) and 7 show that rhenium binds to lactoferrin, due to theincrease in the 231 nm band upon rhenium binding.

FIGS. 6(A), (B) and (C) represent the reaction of apo-lactoferrin withRe/NTA. 1×10⁻⁵M apo-lactoferrin was added to 5 mM sodium bicarbonate(black) and then 5-fold excess Re/NTA was added after 0 mins, 15, 30,90, 180, 240, 300 and 420 minutes. FIG. 6(B) shows the absorption peakat 231 nm, while FIG. 6(C) shows the absorption peak at 500 nm in moredetail. X on FIG. 6(C) indicates the absorption peak at 0 and 15minutes. Y on FIG. 6(C) indicates the shift in the maximum absorptionwavelength at the end of the time course.

FIG. 7 represents the reaction of apo-lactoferrin and sodium bicarbonatewith excess Re/NTA. This reaction was monitored by measuring absorbanceat 231 nm with time.

Both FIGS. 6(A), 6(B), 6(C) and 7 show that rhenium binds tolactoferrin, due to the increase in the 231 nm band upon rheniumbinding.

We believe these experiments show that rhenium can be inserted into thebinding site of transferrin, giving stable complexes within about 1 hourof addition. This chemistry is directly applicable to technetiumchemistry with transferrin and lactoferrin, and by analogy—since thechemistry of technetium and rhenium are so similar—the same chemicalprotocol for rhenium binding to transferrin and lactoferrin may be usedfor technetium binding to the same proteins. The technetium experiments,because of the radioactive nature of technetium cannot be analysed inthe same way. The experiments with technetium binding to transferrin andlactoferrin show directly the uptake of these species into tumour cells.These experiments are described below.

Example 2 Labelling of Transferrin with Technetium^(99m) and Uptake ofTf-Tc Complex by Cancer Cells

Experimental Procedure:

Stock Solutions

The following solutions were made up.

Pertechnetate Solution (^(99m)Tc) [TcO₄ ⁻]:

270 MBq received in 1 ml aqueous solution. (Pertechnetate concentration:5×10⁻¹² g/MBq (3.06×10⁻¹⁴ mol/MBq)) 50 MBq in 0.2 ml (1.53×10⁻¹² M)

Apotransferrin solution (Tf):

Washed 3× with 0.1 M KCl. Final volume=1.2 ml Absorbance=0.65.(Extinction coefficient at 280 nm=93,000 M⁻¹ cm⁻¹). Total Tf=8.4×10⁻⁹mol. Diluted in 5 ml. 0.025 ml diluted to 25 ml (1.68×10⁻¹² mol/ml)

Tin (reducing) Solutions:

SnCl₂.2H₂O 34×10⁻³ g+NaHCO₃ 4.4×10⁻³ g+Fe(NO₃)₃ 12.5×10⁻³ g in 25 ml:Working ion mixtures: 0.25 ml diluted to 25 ml and 0.025 ml of thisdiluted to 25 ml.

Solution Combinations

Following the Re chemistry described previously, the followingcombinations of solutions were mixed at room temperature for 1 h afterwhich 10 ml of PBS was added and the solution mixed. 9 ml was thendiscarded and the remaining 1 ml diluted to 20 ml with PBS.

Solution: 1) 0.2 ml ^(99m)Tc[TcO₄ ⁻] solution+0.025 ml of tin solution.

-   -   2) 0.2 ml ^(99m)Tc[TcO₄ ⁻] solution+0.025 ml of tin solution+0.2        ml Tf.

Example 3

Uptake of Solutions by Cancer Cells

Each solution from Example 2 was incubated with cells as follows. Cellculture: RT112 cells seeded into four 25 cm flasks were grown toconfluency in Dulbecco's Minimum Essential Medium +5% foetal bovineserum. 2 h prior to uptake studies the cells were washed once with Hanksbalanced salt solution (HBSS) and incubated for 2 h at room temperaturewith 4 ml of HBSS. The cells were then washed with PBS and incubated for1.5 h at room temperature with 4 ml of the corresponding (1–4) labelledincubation solution. The incubation solution was then discarded and thecells washed 5 × with PBS, treated with 2 ml of trypsin and 1 ml countedon a Packard gamma counter. A further flask was trypsinized to estimatecell number. Each solution was counted independently and the resultsaveraged to give a measure of uptake.

Results

In each case the Tc is taken-up by the tumour cells. Since the cellsshow phagocytotic behaviour, some non-specific uptake is expected.However, as FIG. 8 shows there is a clear enhancement of Tc uptake (byalmost 50%) once it is complexed to the Tf.

Example 4

Preparation of Product

A product comprising the following components is made up as alyophilised, pyrogen free powder:

COMPOUND AMOUNT mg Lactoferrin 40.0 Tin(II)chloride 0.24 Gentisic acid0.84 Sodium chloride 30

The mixture is then placed in a vial and sealed. The mixture can then bestored at room temperature until required for use.

Example 5

Labelling Lactoferrin

The pertechnetate source is either sodium pertechnetate (^(99m)Tc)injection (Fission) or sodium pertechnetate (^(99m)Tc) (Non-Fission),maximum 7.4 GBq 200 mCi. An amount of the pertechnate is asepticallyadded to a volume of 3–6 ml aqueous solution in a vial and shaken andleft for several minutes (2–5 minutes).

To prepare a dose for a single patient, the vial containing thelactoferrin and other additives is reconstituted in 3–6 ml salinesolution and shaken for 2–5 minutes so as to allow the lyophilisedmaterial to reconstitute in solution. All but approximately 1 ml of thesolution is withdrawn and added to the required amount of sodiumpertechnetate (^(99m)Tc) injection (Fission) or sodium pertechnetate(^(99m)Tc) (Non-Fission). The mixture is then shaken to ensure a highlabelling yield of lactoferrin.

Example 6

Detecting Tumour Site

A single dose of the ^(99m)Tc labelled lactoferrin complex isadministered by the i.v route and γ radiation is observed byconventional means over a selected period of time. The ^(99m)Tc is foundto concentrate in specific areas within the body, the area is found tocorrespond to a tumour site.

1. A composition comprising lactoferrin and a reducing agent forconverting Tc as the pertechnetate (TcO₄ ⁻) to Tc³⁺, wherein thelactoferrin is a serum lactoferrin.
 2. A composition comprisinglactoferrin and a reducing agent for converting To as the pertechnetate(TcO₄ ⁻) to Tc³⁺, which is in unit form and wherein the amount oflactoferrin per unit is in the range of 2–60 mg.
 3. The compositionaccording to claim 1 which is in unit form and wherein the amount oflactoferrin per unit is in the range of 2–60 mg.
 4. A compositioncomprising lactoferrin and a reducing agent for converting Tc as thepertechnetate (TcO₄ ⁻) to Tc³⁺, which is in unit form and wherein thelactoferrin is derived from mammalian tissue or blood and the amount oflactoferrin per unit is in the range of 2–60 mg.
 5. A compositioncomprising lactoferrin and a reducing agent for converting Tc as thepertechnetate (TcO₄ ⁻) to Tc³⁺, which is in unit form and wherein thelactoferrin is a recombinant protein and the amount of lactoferrin perunit is in the range of 2–60 mg.
 6. The composition according to claim1, further comprising a ^(99m)Tc source.
 7. A composition comprisinglactoferrin and a reducing agent for converting Tc as the pertechnetate(TcO₄ ⁻) to Tc³⁺, wherein the lactoferrin is a recombinant protein, andfurther comprising a ^(99m)Tc source.
 8. The composition according toclaim 6, wherein the ^(99m)Tc source is pertechnetate TcO_(4.)
 9. Thecomposition according to claim 7, wherein the ^(99m)Tc source ispertechnetate TcO_(4.)
 10. A composition comprising lactoferrin and areducing agent for converting Tc as the pertechnetate (TcO₄ ⁻) to Tc³⁻,further comprising a ^(99m)Tc source, wherein the ^(99m)Tc source isprovided separately from the lactoferrin and reducing agent.
 11. Acomposition comprising lactoferrin and a reducing agent for convertingTc as the pertechnetate (TcO₄ ⁻) to Tc³⁻, further comprising a ^(99m)Tcsource, wherein the amount of ^(99m)Tc is about 7.4 GBq (200 mCi).
 12. Acomposition comprising lactoferrin and a reducing agent for convertingTc as the pertechnetate (TcO₄ ⁻) to Tc³⁻, and further comprising asolubilizing agent and/or an isotonic agent.
 13. The compositionaccording to claim 12, wherein the solubilizing agent is gentisic acid.14. The composition according to claim 12 which is in unit form andwherein the amount of solubilizing agent per unit is in the range ofabout 0.7 mg. to about 0.9 mg.
 15. The composition according to claim12, wherein the isotonic agent is sodium chloride.
 16. The compositionaccording to claim 12 which is in unit form and wherein the amount ofisotonic agent per unit is in the range of about 20 mg to about 40 mg.17. A composition comprising lactoferrin and a reducing agent forconverting Tc as the pertechnetate (TcO₄ ⁻) to Tc³⁻, wherein thecomposition is lyophilized.
 18. A composition comprising lactoferrin anda reducing agent for converting Tc as the pertechnetate (TcO₄ ⁻) toTc³⁻, wherein the composition is in powder form.
 19. A kit comprising afirst composition comprising the lactoferrin and a reducing agent forconverting Tc as the pertechnetate (TcO₄ ⁻) to Tc³⁻ in a first sealedvessel and a second composition comprising a radionuclide labeledlactoferrin complex in a second sealed vessel and optionally, a set ofwritten instructions.